Amplification of DNA by polymerase chain reaction (PCR) is a technique fundamental to molecular biology. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real time” PCR. See, for example, U.S. Pat. No. 6,174,670, incorporated herein by reference.
Monitoring fluorescence each cycle of PCR initially involved the use of ethidium bromide. Higuchi R, G Dollinger, P S Walsh and R. Griffith, Simultaneous amplification and detection of specific DNA sequences, Bio/Technology 10:413-417, 1992; Higuchi R, C Fockler G Dollinger and R Watson, Kinetic PCR analysis: real time monitoring of DNA amplification reactions, Bio/Technology 11: 1026-1030, 1993. In that system fluorescence is measured once per cycle as a relative measure of product concentration. Ethidium bromide detects double stranded DNA; if template is present fluorescence intensity increases with temperature cycling. Furthermore, the cycle number where an increase in fluorescence is first detected increases inversely proportionally to the log of the initial template concentration. Other fluorescent systems have been developed that are capable of providing additional data concerning the nucleic acid concentration and sequence.
While PCR is an invaluable molecular biology tool, the practical implementation of real time PCR techniques has lagged behind the conceptual promise. Currently available instrumentation generally does not actually analyze data during PCR; it simply acquires the data for later analysis. After PCR has been completed, multiple manual steps are necessary to analyze the acquired data, and human judgment is typically required to provide the analysis result. What is needed is a system for automating data acquisition and analysis so that no user intervention is required for reporting the analytical results. Thus, when the temperature cycling in a polymerase chain reaction amplification is complete, the system software is automatically triggered and the results, for example, the presence or absence of a given pathogen, are immediately displayed on screen. Algorithms for detection, quantification, and genotyping are needed. Moreover, initiation of the analysis algorithm can be implemented prior to completion of temperature cycling. Data processing can occur during amplification and concomitant analysis results can be used to modify temperature cycling and to acquire additional data during the latter stages of the amplification procedure to optimize amplification protocol and data quality.
A major problem in automating PCR data analysis is identification of baseline fluorescence. Background fluorescence varies from reaction to reaction. Moreover, baseline drift, wherein fluorescence increases or decreases without relation to amplification of nucleic acids in the sample, is a common occurrence. Prior attempts to automate amplification data analysis involved setting the baseline fluorescence as that measured at one or more predetermined early cycle numbers. This technique accounts for the variation in background fluorescence, but it does not compensate for baseline drift. Without compensation for baseline drift, automated amplification data analysis can easily provide both false negative and false positive results.
Thus, a method of determining the presence of a nucleic acid in a sample is provided, the method comprising the steps of providing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a signal related to the quantity of the nucleic acid, amplifying the nucleic acid through a plurality of amplification cycles in the presence of the fluorescent entity, measuring fluorescence intensity of the fluorescent entity at each of the plurality of amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle, obtaining a score from each of a plurality of tests, each of the plurality of tests using the fluorescence values to generate the score, and using the scores to ascertain whether the nucleic acid is present in the sample. In an illustrated embodiment, the tests comprise a Confidence Interval Test, and a Signal-to-Noise-Ratio Test.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.